Competition experiments, employing 14C-labeled samples of glycerol and glycolaldehyde, indicate that in Escherichia coli B there are two independent pathways leading to pyridoxal. In mutant WG2 (and therefore presumably also in the wild strain) the major pathway utilizes glycerol and related trioses as the sole carbon source in the construction of the C8N skeleton of pyridoxol: C-1, -3 of glycerol yields C-2', -3, -4', -5' and -6, C-2 of glycerol yields C-2, -4, and -5 of the vitamin. In the minor pathway glycolaldehyde and not glycerol supplies C-5 and C-5' of pyridoxol, while glycerol is the source of the other 6 carbon atoms. In mutant WG3 the major route is blocked and the "glycolaldehyde pathway" becomes the sole source of vitamin B6.